Regulation of the Ste20-like kinase, SLK: involvement of activation segment phosphorylation

Expression and activation of the Ste20-like kinase, SLK, is increased during kidney development and recovery from ischemic acute kidney injury. SLK promotes apoptosis, and it may regulate cell survival during injury or repair. This study addresses the role of phosphorylation in the regulation of kinase activity. We mutated serine and threonine residues in the putative activation segment of the SLK catalytic domain and expressed wild type (WT) and mutant proteins in COS-1 or glomerular epithelial cells. Compared with SLK WT, the T183A, S189A, and T183A/S189A mutants showed reduced in vitro kinase activity. SLK WT, but not mutants, increased activation-specific phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. Similarly, SLK WT stimulated activator protein-1 reporter activity, but activation of activator protein-1 by the three SLK mutants was ineffective. To test if homodimerization of SLK affects phosphorylation, the cDNA encoding SLK amino acids 1-373 (which include the catalytic domain) was fused with a cDNA for a modified FK506-binding protein, Fv (Fv-SLK 1-373). After transfection, the addition of AP20187 (an FK506 analog) induced regulated dimerization of Fv-SLK 1-373. AP20187-stimulated dimerization enhanced the kinase activity of Fv-SLK 1-373 WT. In contrast, kinase activity of Fv-SLK 1-373 T183A/S189A was weak and was not enhanced after dimerization. Finally, apoptosis was increased after expression of Fv-SLK 1-373 WT but not T183A/S189A. Thus, phosphorylation of Thr-183 and Ser-189 plays a key role in the activation and signaling of SLK and could represent a target for novel therapeutic approaches to renal injury.     

Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.     

Absence of Dpy19l2, a new inner nuclear membrane protein, causes globozoospermia in mice by preventing the anchoring of the acrosome to the nucleus

Sperm-head elongation and acrosome formation, which take place during the last stages of spermatogenesis, are essential to produce competent spermatozoa that are able to cross the oocyte zona pellucida and to achieve fertilization. During acrosome biogenesis, acrosome attachment and spreading over the nucleus are still poorly understood and to date no proteins have been described to link the acrosome to the nucleus. We recently demonstrated that a deletion of DPY19L2, a gene coding for an uncharacterized protein, was responsible for a majority of cases of type I globozoospermia, a rare cause of male infertility that is characterized by the exclusive production of round-headed acrosomeless spermatozoa. Here, using Dpy19l2 knockout mice, we describe the cellular function of the Dpy19l2 protein. We demonstrate that the protein is expressed predominantly in spermatids with a very specific localization restricted to the inner nuclear membrane facing the acrosomal vesicle. We show that the absence of Dpy19l2 leads to the destabilization of both the nuclear dense lamina (NDL) and the junction between the acroplaxome and the nuclear envelope. Consequently, the acrosome and the manchette fail to be linked to the nucleus leading to the disruption of vesicular trafficking, failure of sperm nuclear shaping and eventually to the elimination of the unbound acrosomal vesicle. Finally, we show for the first time that Dpy19l3 proteins are also located in the inner nuclear envelope, therefore implying that the Dpy19 proteins constitute a new family of structural transmembrane proteins of the nuclear envelope.     

Intrauterine growth restriction leads to changes in sulfur amino acid metabolism, but not global DNA methylation, in Yucatan miniature piglets

Intrauterine growth restriction (IUGR), in both animals and humans, has been linked to metabolic syndrome later in life. There has been recent evidence that perturbations in sulfur amino acid metabolism may be involved in this early programming phenomenon. Methionine is the precursor for cellular methylation reactions and for the synthesis of cysteine. It has been suggested that the mechanism behind the "fetal origins" of adult diseases may be epigenetic, involving DNA methylation. Because we have recently demonstrated the fetal origins phenomenon in Yucatan miniature swine, we hypothesized that sulfur amino acid metabolism is altered in IUGR piglets. In this study, metabolites and the activities of sulfur amino acid cycle enzymes were analyzed in liver samples of 3- to 5-day-old runt (IUGR: 0.85±0.13 kg) and large (1.36±0.21 kg) Yucatan miniature pig littermates (n=6 pairs). The IUGR piglets had significantly lower specific and total activities of betaine-homocysteine methyltransferase (BHMT) and cystathionine γ-lyase (CGL) than larger littermates (P<.05). Expression of CGL (but not BHMT) mRNA was also lower in IUGR piglets (P<.05). This low CGL reduced cysteine and taurine concentrations in IUGR pigs and led to an accumulation of hepatic cystathionine, with lower homocysteine concentrations. Methylation index and liver global DNA methylation were unaltered. Reduced prenatal growth in Yucatan miniature piglets impairs their remethylation capacity as well as their ability to remove cystathionine and synthesize cysteine and taurine, which could have important implications on long-term health outcomes of IUGR neonates.     

Rapid climate change and the rate of adaptation: insight from experimental quantitative genetics

CONTENTS: Summary 752 I. Introduction 752 II. Will migration be enough? 753 III. Can adaptation proceed fast enough? 754 IV. Fitness links demographic and evolutionary processes 755 V. Experimental studies: what do they tell us and how can we improve them? 756 VI. Predicting evolutionary change based on genetic variation and natural selection 757 VII. The chronosequence approach 758 VIII. Resurrection of ancestral propagules 759 IX. The mean and variance in fitness, a link between genetics and demography 760 X. Conclusions 762 Acknowledgements 762 References 762 SUMMARY: Evolution proceeds unceasingly in all biological populations. It is clear that climate-driven evolution has molded plants in deep time and within extant populations. However, it is less certain whether adaptive evolution can proceed sufficiently rapidly to maintain the fitness and demographic stability of populations subjected to exceptionally rapid contemporary climate change. Here, we consider this question, drawing on current evidence on the rate of plant range shifts and the potential for an adaptive evolutionary response. We emphasize advances in understanding based on theoretical studies that model interacting evolutionary processes, and we provide an overview of quantitative genetic approaches that can parameterize these models to provide more meaningful predictions of the dynamic interplay between genetics, demography and evolution. We outline further research that can clarify both the adaptive potential of plant populations as climate continues to change and the role played by ongoing adaptation in their persistence.     

The B gene of pea encodes a defective flavonoid 3',5'-hydroxylase, and confers pink flower color

The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5' position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the enzyme that hydroxylates the 5' position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3'5'H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3'5'H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3'5'H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.